lab (4) western blotfaculty.ksu.edu.sa/sites/default/files/lab_4_24.pdf · §the color/florescence...
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Lab (4) Western Blot
BCH 462- Biotechnology & Genetic engineering [Practical]
Immunoassay§ Immunoassay:
A test that uses highly specific and selective antigen-antibody reactions forming antibody and antigen
complexes [immuno-complexes] as a means of generating measurable results.
§ Antigens [Ag]:
A substance that when introduced into the body stimulates the production of an antibody.
Antigens include toxins, bacteria, foreign blood cells, and the cells of transplanted organs.
§ Antibody [Ab]:
Antibodies are large Y-shaped glycoproteins. They are produced by the immune system to identify and neutralize
foreign objects (antigens).
Antibody and antigen complexes [immuno-complexes]
💡 Remember !!Antigen = Protein of interestImmunoassay
Blotting
§ Blotting is a method of transferring proteins, DNA or RNA, onto a carrier (for instance, a
nitrocellulose, PVDF or nylon membrane).
§ Types:
1. Southern blotting; used to detect DNA.
2. Northern blotting; used to detect RNA.
3. Eastern blotting; used to analyze protein post translational modifications (PTM).
4. Western blotting; used to detect Proteins.
Overview of blotting technique
Western blot
§ Also called protein immunoblot.
§ It is a widely used immunoassay technique.
§ Used to identify specific proteins [antigens] in a sample of tissue homogenate or extract, based
on their ability [the antigens] to bind to antibodies resulting in color/florescence indicate the
presence of this specific protein.
Applications:
1. Analyzing, identifying target proteins and estimating their molecular weight.
2. To compare the amounts of a protein of interest among different samples.
3. Used in clinical laboratories for assisting identification of certain antigen proteins (pathogen or biomarker).
4. Used to detect changes in protein expression under different biological conditions (e.g. in disease, stress,
etc.).
Practical Part 🧬
Practical part
• To understand how proteins (antigens) can be analyzed using antibodies raised against these
proteins by immunoblotting technique.
• To perform the steps of western-blot technique to detect tubulin protein.
§ Aims:
Practical part
§ The mixture of proteins is separated based on molecular weight.
§ These results are then electro-transferred to solid support producing a band for each protein.
§ The transferred protein is detected by incubating the gel with specific primary antibody to the protein of
interest, secondary antibody labelled with an enzyme or fluorophore which target the primary
antibody, and substrate which in the end you will get colored product.
§ The color/florescence indicates the presence of the protein of interest.
§ The thickness of the band corresponds to the amount of protein present.
§ Thus, the molecular weight and amount of the desired protein can be characterized from a complex mixture of
proteins by western blotting.
Western blot performing steps The technique uses three elements to accomplish this task
1. 1st phase (SDS-PAGE):
Separation sample mixture by size using SDS-PAGE.
2. 2nd phase (Electro-blotting):
Transfer to a solid support (electro-blotting) by transferring the
proteins bands from the gel to the membrane.
3. 3rd phase (Marking target protein to visualize):
Marking target protein using a proper primary and secondary
antibody to visualize.
1st phase (SDS-PAGE)
§ A protein sample is subjected to polyacrylamide gel electrophoresis.
§ To confirm the separation of samples (since separated proteins are colorless) use:
1. Replica of the gel and stain it as usual [with Coomassie brilliant blue R-250].
2. Using a pre-stained marker.
3. Reversible staining by Ponceau S.
Pre-stained marker
Protein Ladder is a mixture of nine (9) blue-, orange- and
green-stained proteins (10 to 250kDa) for use as size
standards in protein electrophoresis (SDS-PAGE) and
Western blotting.
Ponceau staining is a washable light red colored dye , that may
be used to prepare a stain for rapid detection of protein bands
on nitrocellulose or polyvinylidene fluoride (PVDF) membranes
(Western blotting).
Ponceau staining
2nd phase (Electro-blotting)§ After that the gel is placed over a sheet of PVDF, the protein in the gel is electrophoretically
transferred to the Polyvinylidene fluoride (PVDF) membrane. “transfer step [Electroblotting]”.
§ Methods of transfer:
1. Wet method → Most common transfer method, best for proteins >100kDa.
2. Semi-wet → Quick but less efficient.
3. Dry → Quicker, No transfer buffer required.
Transfer sandwich
§ Because the samples in the gel are [–ve] charged , the applied electric current will facilitate their
transferring to nitrocellulose membrane, the samples will move toward the Anode[ +].
§ Also the capillary action has its effect in the movement of the samples from the gel to the
nitrocellulose membrane.
§ Note that: [the filter papers, gel and PVDF membrane will be soaked in transfer buffer].
3rd phase (Marking target protein to visualize)§ The PVDF is then soaked in blocking buffer.
§ The PVDF is then incubated with the specific primary antibody for the protein of interest.
3rd phase (Marking target protein to visualize)
§ The PVDF is then washed and incubated with a secondary antibody, which is specific for
the first antibody [primary–antibody].
• The color produced indicate the presence of the antibody-antigen [Ab-Ag] complex.
3rd phase (Marking target protein to visualize)
Types of western blot detection methods “detection step”:
1- Colorimetric
Secondary antibody labeled with enzyme conjugates, such as alkaline phosphatase (AP) or horseradish
peroxidase (HRP), when provided with a chromogenic substrate, will cause a color reaction.
2- Chemiluminescence
The enzyme attached to the secondary antibody triggers a reaction with a luminescent substrate that produces
light as a by-product.
3- Fluorescence
Uses secondary antibodies that are conjugated to specific fluorophores that can absorb and emit light within a
range of wavelengths, so no additional substrate is necessary.
Western blot detection methods:
¡ Thus the molecular weight and amount of the desired protein can be characterized from a complex mixture of proteins by western blotting.
Detection of specific protein using Western blot
Performing western blot:
http://www.youtube.com/watch?v=VgAuZ6dBOfs
Supporting materials: